Project Summary Type I diabetes (T1D) is a T cell-mediated autoimmune disease which is dramatically increasing in incidence. The pathogenesis of T1D is complex and incompletely understood, involving progressive destruction of pancreatic insulin-producing b cells by CD8 T cells, which leads to insulin deficiency and loss of glucose homeostasis. However, many aspects of the programming and regulation of self-reactive CD8 T cells mediating autoimmunity remain enigmatic. Utilizing the clinically relevant non-obese diabetic (NOD) mouse model of T1D, I investigated the activation and differentiation of b cell antigen-specific CD8 T cells throughout the course of T1D. I found that antigen-specific CD8 T cells exhibited phenotypic heterogeneity in the pancreatic draining lymph node (pLN) and in the pancreas. The specific hypothesis of this proposal is that the pancreatic autoimmune CD8 T cell response in T1D is driven by heterogeneous populations arising in the pLN. In this study, I will define the spatiotemporal factors that determine CD8 T cell population heterogeneity and identify transcription factor networks that define distinct autoimmune T cell populations in the pLN and pancreas in T1D. High-throughput RNA sequencing, in vivo gain and loss of function studies, and pharmacologic modulation experiments will be performed to determine the phenotypic, functional, and molecular characteristics of antigen- specific autoimmune CD8 T cells. Single-cell RNA sequencing will be employed to define population heterogeneity. This proposal is tailored for a physician-scientist in training, as it investigates the molecular mechanisms underlying a human disease and may identify promising molecular targets for the prevention or treatment of T1D and other T cell-mediated autoimmune diseases.